Proc. Natl. Acad. Sci. USA Vol. 74, No. 3, pp. 864-868, March 1977 Biochemistry Isopeptide linkage between nonhistone and histone 2A polypeptides of chromosomal conjugate-protein A24 (chromatin/amino acid sequence/tryptic peptides) IRA L. GOLDKNOPF AND HARRIS BUSCH Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030 Communicated by Stanford Moore, November 29,1976 ABSTRACT Chromosomal protein A24 has a unique The present study indicates that protein A24 has two amino structure inasmuch as it contains histone 2A and a nonhistone termini and one carboxyl terminus. In addition, the amino acid polypeptide the sequence of which has been partially deter- mined. Comparative analysis of the ninhydrin-insensitive sequence of the branched tryptic peptide 17' indicates that the amino-terminal tryptic peptides of protein A24 and histone 2A eamino group of lysine 119 of the histone 2A portion of protein and a quantitative analysis of their carboxyl-terminal amino A24 is linked to glycine by an isopeptide bond. Therefore, a acid indicated that protein A24 has two amino termini and one branched structure is proposed for protein A24. carboxyl terminus. The amino acid sequence analysis of tryptic peptide 17' of protein A24: MATERIALS AND METHODS H2N-CH4N-CHNH Protein Isolation. Protein A24 and histone 2A, isolated from (Gly) Y)(lY(I H240 calf thymus tissue as described (3, 7), were obtained in highly HN-CH-C;-Thr-Gkj-Ser-H6*is-Lys purified form, as determined by two-dimensional polyacryl- (Lys 119) amide gel electrophoresis (8). showed it contains tryptic peptide 17 of histone 2A, Lys-Thr- Preparation of Tryptic Peptides. Protein A24 and histone Glu-Ser-His-His-Lys. Lysine 119, the amino terminus of this 2A (4.5 mg each) were simultaneously subjected to two diges- peptide, which is derived from the histone 2A portion of protein tions for 2 hr each with 1% by weight of trypsin as described A24, is linked by an isopeptide bond to the carboxyl group of a (3, 4). Aliquots of each protein sample were also hydrolyzed and glycine residue. Accordingly, the branched structure of protein subjected to amino acid analysis as described (9) to determine A24 proposed is: yields of individual peptides. The digests were then subjected H OH to chromatography in butanol:acetic acid:water, 4:1:5 (vol/vol) Met-- -4-CH:-84J-CHj-NH (10) followed by electrophoresis in pyridine:acetic acid:water, (Gly) (Gly) (CI ) 1:10:189 (vol/vol), pH 3.6 (10). The separations were monitored ",I t.0 N-AcetyIserine -------CH-C- --Lys by staining with Sakaguchi reagent (11), fluorescamine (12), Histone 2A: I Lys 119 129 ninhydrin cadmium, or starch iodide reagents (11). Separated peptides were eluted from the paper with 30% acetic acid as Initial interest in chromosomal protein A24 arose from a described (11). marked reduction in its nucleolar levels during the liver nu- Quantitative Hydrazinolysis. Protein A24 and histone 2A cleolar hypertrophy and increased rRNA synthesis induced (0.5 mg each, quantitated by amino acid analysis as noted either by the administration of thioacetamide (1) or by partial above) were subjected to hydrazinolysis at 850 for 28 hr to hepatectomy (2). Isolation and initial chemical characterization minimize destruction of lysine as described (13). For determi- indicated that protein A24 was a nonhistone chromosomal nation of carboxyl-terminal amino acids, the hydrazinolysates protein with approximately equal amounts of acidic and basic were extracted with benzaldehyde to remove hydrazides and amino acids; it constituted approximately 1.9% of the sum of the free amino acids were analyzed by amino acid analysis. histones 2A, 2B, 3, and 4 (3). Protein A24 was found to contain Purified peptides were subjected to hydrazinolysis in the same the tryptic and chymotryptic peptides of histone 2A as well as fashion. Peptides containing acetyl groups yielded acetyl hy- additional peptides and, accordingly, it was suggested that drazide upon hydrazinolysis. Accordingly, to analyze for the protein A24 contained a "nonhistone-like" polypeptide linked presence of acetyl hydrazide, we subjected the hydrazinolysates to a histone 2A molecule (4). More recently (5, 6), protein A24 to chromatography in collidine:H20,10:2 (vol/vol) (14). Hy- has been shown to contain the amino-terminal sequence: drazides were detected by the staining method of Andrae (15). Standard acetyl hydrazide was prepared by hydrazinolysis of 1 5 10 acetyl valine (Schwarz/Mann). Met-Gln- Ile -Phe- Val-Lys-Thr-Leu-Thr-Gly- Edman Degradation. The amino acid sequences of peptides 11 15 20 were determined by a modification of the Edman degradation Lys-Thr- Ile -Thr-Leu-Glu-Val-Glu-Pro-Ser - (16, 17). For analysis of Edman degradation products, thiazo- 21 25 30 linones were either hydrolyzed under reduced pressure in hy- Asp-Thr- Ile -Glu-Asn-Val-Lys-Ala-Lys-Ile drogen iodide (18) and subjected to amino acid analysis, or they 31 35 were first converted to phenylthiohydantoins in 1 M HCl at 80° Gln -Asp-Lys-Glu-Gly- Ile -Pro- for 10 min, extracted in ethyl acetate, and analyzed by gas This sequence is not homologous to histone sequences. On the chromatography (19). The amounts of amino acids released other hand, the carboxyl-terminal amino acid sequence of from thiazolinones were corrected for destruction during HI protein A24 was identical to that of histone 2A (5, 6). hydrolysis as described (5). 864 Downloaded by guest on September 25, 2021 Biochemistry: Goldknopf and Busch Proc. Natl. Acad. Sci. USA 74 (1977) 865 A 2A 4.q#vHISTONE *sqt410. .Mi.k3 6 5 15 4 p i 4 14 4 .-% p 1 7 9 4:.. c Protein A24 -.12 _11 .4 -13 8.,6 5 15 P4 Al ,14 8 9.6 -.0-171~~ FIG. 1. Two-dimensional peptide maps and c: rresponding diagrams of (A and B) histone 2A and (C and D) protein A24 tryptic digests. Filled spots are common to both proteins. Note the positions of peptides 16 (diagrams), which were negative to the ninhydrin cadmium stain used in the maps. Note also the position of peptides 17 of histone 2A and 17' of protein A24, which differ slightly in electrophoretic mobility. RESULTS In the present study a quantitative analysis of carboxyl-terminal The two amino termini of protein A24 amino acids was undertaken to determine if protein A24 had an additional carboxyl terminus as well. Quantitative hydraz- As reported previously, a comparison of tryptic peptide maps inolysis released molar yields of carboxyl-terminal lysine of 1.01 of histone 2A and protein A24 revealed remarkable similarities and 0.88 for protein A24 and histone 2A, respectively;* no other (4) as indicated in Fig. 1. However, the peptide designated 16 carboxyl-terminal amino acids were detected in protein A24. (diagrams, Fig. 1) was not previously detected in protein A24 because it did not stain with the ninhydrin-cadmium (peptide The branched tryptic peptide of protein A24 maps, Fig. 1) or fluorescamine procedures, which require the The detection of two amino termini and one carboxyl terminus presence of a primary amino group (Table 1). This peptide had suggested that the protein A24 molecule was branched and that been reported to be present in histone 2A (7) and in fact con- the nonhistone polypeptide was linked to histone 2A in a tained the blocked amino terminus of the histone (20, 21). Ac- manner that prevented detection of its carboxyl terminus. A cordingly, an analysis was performed of purified peptides 16 search was made for an altered tryptic peptide of protein A24. from histone 2A and protein A24 (Table 1). Both peptides had Although the other peptides had similar electrophoretic identical staining characteristics, amino acid composition, and mobilities, tryptic peptide 17' from protein A24 had a slightly positive tests for the acetyl group. This was consistent with the different electrophoretic mobility from peptide 17 of histone structure previously determined (20, 21) for the blocked 2A (Fig. 1). Accordingly, these peptides were subjected to amino-terminal tryptic peptide of histone 2A: amino acid analysis, quantitative hydrazinolysis, and sequential N-Acetylserine-Gly-Arg. Edman degradation. The amino acid composition and carboxyl terminus of pep- Thus, in addition, to the nonhistone-like free amino-terminal tide 17 of histone 2A (Table 2) were found to be the same as that amino acid sequence previously detected (3, 5, 6), protein A24 reported previously (22) for a peptide containing amino acid also contained the blocked amino terminus of histone 2A. residue 119-125 of the histone 2A sequence (23). Edman deg- radation confirmed the amino The (Table 3) identity and acid se- single carboxyl terminus of protein A24 quence of this peptide (Fig. 2A). Carboxypeptidase A and B digestion indicated that protein A24 The amino acid composition of peptide 17' of protein A24 contains the carboxyl-terminal sequence (5, 6) identical to that (Table 2) was the same as that of peptide 17 of histone 2A except of histone 2A (22, 23): for the presence of two additional glycine residues. The yield -His -His -Lys -Ala -Lys -Gly -Lys * The molar yield calculations were based on the molecular 123 weights 124 125 126 127 128 129 of 27,000 and 14,000 for protein A24 (3) and histone 2A (23), re- (histone 2A residue number) spectively. Downloaded by guest on September 25, 2021 866 Biochemistry: Goldknopf and Busch Proc. Nati. Acad. Sci. USA 74 (1977) Table 1. Analysis of tryptic peptide 16 of protein A24 Table 3. Sequential Edman degradation of peptides 17 and and histone 2A 17' (nmol of amino acids per nmol of peptide released from thiazolinones after hydrolysis with HI) Protein A24 Histone 2A Edman cycle Staining reactions: Amino Fluorescamine - - acid 1 2 3 4 Ninhydrin Sakaguchi + + Peptide 17 of histone 2A Rydon-Smith + + Gly Amino acid ratios: Lys 0.5 Ser (= 1.00) 1.00 1.00 Thr 1.0 Gly 1.09 1.15 Glu 0.9 Arg 1.12 1.21 Ser 0.3 Molar yield* 0.42 0.46 Peptide 17' of protein A24 Acetyl groupt + + Gly 1.6 0.5 * Nanomoles recovered from tryptic peptide maps per nanomole of Lys 0.4 0.1 protein digested.